Acetogenin-induced fibrotic heart disease from avocado (Persea americana, Lauraceae) poisoning in horses.

Poisoning by avocado (Persea americana) has been confirmed in sheep, goats, dogs, rabbits and ostriches. The clinical signs and lesions are attributed to the acetogenin, persin. Little is known regarding the epidemiology, clinical signs, lesions and therapy caused by acetogenin-induced heart damage. During the two-year study, we investigated a horse farm with six horses that often fed themselves with P. americana leaves or mature fruit pulp and skin on the ground. Two horses died, and one underwent necropsy, histopathology, and immunohistochemistry using the anti-cardiac troponin C (cTnC). Grossly and histopathologically, there was severe cardiac fibroplasia. Immunohistochemically, there was a multifocal decrease or negative expression in the cTnC cardiomyocytes' cytoplasm. Persea americana leaves were confirmed in the alimentary tract using botanical anatomy and molecular techniques. The chemical investigation by (LC-ESI-MS) revealed the presence of the acetogenins, persin and avocadene 1-acetate from P. americana. Persin was present in leaves and fruits (seed and pulp), while avocadene 1-acetate was found in leaves and fruits (seed, peel, and pulp) with a higher concentration in the pulp. Four other horses have been examined by electrocardiogram, echocardiogram and serum Troponin 1 (cTnI). To establish a causal effect of consumption of P. Americana and heart fibroplasia in horses, long-time experiments must be carried out.

Toxicities of acetogenin-based bioacaricides against two-spotted spider mite and selectivity to its phytoseiid predators.

Tetranychus urticae is the main pest of strawberry crops and can cause up to 80% of productivity losses under high infestations. Aiming to search T. urticae management alternatives compatible with eco-friendly or organic-based food production systems, this study evaluated the lethal and sublethal toxicities of formulated derivatives from Annonaceae (rich in acetogenins) against this pest species. In addition, it also evaluated the selectivity of the most promising formulation to the predatory mites Neoseiulus californicus and Phytoseiulus macropilis, which are largely applied in biological control in Brazil. Among the derivatives tested, the emulsion from the ethanolic seed extract of Annona mucosa-ESEAm (major component: acetogenin bis-tetrahydrofuran rolliniastatin-1) caused pronounced mortality of T. urticae after 120 h of exposure (LC50 = 465.5 mg L-1), in a comparable or superior manner to an abamectin-based synthetic acaricide used as positive control (LC50 = 1243.4 mg L-1). Moreover, ESEAm exposure resulted in a significant decrease in the number of eggs laid by females and caused the most pronounced ovicidal action for T. urticae, with only 5% embryonic viability. However, ESEAm also showed high toxicity to the predatory mites tested, causing 100% mortality for both species after 120 h exposure, similar to abamectin. The interaction between these bioacaricides and biological control agents should be tested under field conditions to further assess the potential ecological selectivity of these derivatives.

Eight new cytotoxic annonaceous acetogenins from the seeds of Annona squamosa.

Eight new annonaceous acetogenins, squamotin A-D (1-4), annosquatin IV-V (5 and 6), muricin O (7) and squamosten B (8), together with four known ones (9-12) were isolated from the seeds of Annona squamosa. Their structures were elucidated by chemical methods and spectral data. The inhibitory activities of compound 1-9 against three multidrug resistance cell lines were evaluated. All tested compounds showed strong cytotoxicity.

Mechanistic Study of Tetrahydrofuran- acetogenins In Triggering Endoplasmic Reticulum Stress Response-apotoposis in Human Nasopharyngeal Carcinoma.

For past three decades, numerous studies have elucidated the antiproliferative effects of acetogenins in hopes of developing a new class of clinical anticancer agents. However, clear and definitive action mechanisms of acetogenins were less clarified. In the present study, three tetrahydrofuran (THF)-containing acetogenins were found to have potent and selective antiproliferative activity against human nasopharyngeal carcinoma (NPC) cell lines and their methotrexate-resistant counterparts. The THF-containing acetogenins induced G2/M phase arrest, mitochondrial damage and apoptosis, and increased cytosolic and mitochondrial Ca2+ in NPCs. Microarray analysis of NPC-TW01 cells treated with squamostatin A, a non-adjacent bis-THF acetogenin, demonstrated an increased endoplasmic reticulum (ER)-stress response (ESR). Enhanced ESR in squamostatin A-treated cells was confirmed by real-time PCR, Western blot and shRNA gene knockdown experiments. Although our results showed that squamostatin A-induced ESR was independent of extracellular Ca2+, the presence of extracellular Ca2+ enhanced the antiproliferative effect of acetogenins. In vivo analyses demonstrated that squamostatin A showed good pharmacokinetic properties and significantly retarded NPC tumor growth in the xenograft mouse model. Conclusively, our work demonstrates that acetogenins are effective and selective inducers of the ESR that can block NPC proliferation, and illustrate a previously unappreciated antitumor mechanism of acetogenins that is effective against nasopharyngeal malignancies.

Four cytotoxic annonaceous acetogenins from the seeds of Annona squamosa.

Four new annonaceous acetogenins (ACGs), squamocin-I (1), II (2) and III (3) and squamoxinone-D (4), together with seven known ACGs (5-11), were isolated from the seeds of Annona squamosa. The structures of all isolates were elucidated and characterised by spectral and chemical methods. Compounds 1-4 were evaluated for their cytotoxicities against Hep G2, SMMC 7721, BEL 7402, BGC 803 and H460 human cancer cell lines. Compound 1 exhibited better potent activity than the positive compound and compound 3 shows selectively cytotoxical activity against H460 with IC50 values of 0.0492 µg/ml.

Non-natural acetogenin analogues as potent Trypanosoma brucei inhibitors.

Neglected tropical diseases remain a serious global health concern. Here, a series of novel bis-tetrahydropyran 1,4-triazole analogues based on the framework of chamuvarinin, a polyketide natural product isolated from the annonaceae plant species are detailed. The analogues synthesized display low micromolar trypanocidal activities towards both bloodstream and insect forms of Trypanosoma brucei, the causative agent of African sleeping sickness, also known as Human African Trypanosomiasis (HAT). A divergent synthetic strategy was adopted for the synthesis of the key tetrahydropyran intermediates to enable rapid access to diastereochemical variation either side of the 1,4-triazole core. The resulting diastereomeric analogues displayed varying degrees of trypanocidal activity and selectivity in structure-activity relationship studies. Together, the biological potency and calculated lipophilicity values indicate that while there is room for improvement, these derivatives may represent a promising novel class of anti-HAT agents.

Synthesis and anti-tumor activity of carbohydrate analogues of the tetrahydrofuran containing acetogenins.

The tetrahydrofuran (THF) containing annonaceous acetogenins (AAs) are attractive candidates for drug development because of their potent cytotoxicity against a wide range of tumors and their relatively simple and robust structures. Replacement of the THF segment with a sugar residue may deliver analogues with improved tumor selectivity and pharmacokinetics and are therefore attractive for drug development. As a first test to the feasibility of such structures, a set of such monosaccharide analogues was synthesized and assayed against four human tumor cell lines, cervical (HeLa), breast (MDA-MB231), T-cell leukemia (Jurkat) and prostate (PC-3). Certain analogues showed low micromolar activity that was comparable to a structurally similar, naturally occurring mono-THF acetogenin. A preliminary examination of the structure-activity profile of these carbohydrate analogues suggests that they have a similar mechanism of action as their THF congeners.

Antitumor activity and toxicity relationship of annonaceous acetogenins.

Annonaceous acetogenins (ACGs) are one of the most interesting classes of natural products appearing in the past two decades. Here, we studied the antitumor activity and toxicity relationship of ACGs including annosquamin B (1), bullatacin (2) and annosquatin B (3) in vivo. A single intraperitoneal (i.p.) injection of 100 µg/kg of annosquamin B, bullatacin and annosquatin B did not cause side effects in normal mice. Bullatacin treatment with five doses of 25 and 50 µg/kg in H22 hepatoma cells bearing mice resulted in about 61% reduction in tumor growth with hematologic parameters increased significantly in normal mice. Annosquamin B and annosquatin B treatments with 10 doses of 25, 50 and 100 µg/kg in the H22 hepatoma cells transplantation tumor model mice resulted in maximum 53.7% and 58.7% reduction in tumor growth, respectively, and did not cause severe side effects in normal mice. This study provided the evidence that adjacent bis-THF ACGs showed higher antitumor activity and toxicity than mono-THF and nonadjacent bis-THF ACGs in vivo. Furthermore, it was found that bullatacin led to liver and kidney toxicity via increasing calcium concentration, ROS production, and Bax expression and Bax/Bcl-2 ratio in rats with repeated treatment with bullatacin for 3 weeks.

Sesquiterpene and acetogenin derivatives from the marine red alga Laurencia okamurai.

In addition to 13 known compounds, four new bisabolane sesquiterpenes, okamurenes A-D (1-4), a new chamigrane derivative, okamurene E (5), and a new C12-acetogenin, okamuragenin (6), were isolated from the marine red alga Laurencia okamurai. The structures of these compounds were determined through detailed spectroscopic analyses. Of these, okamurenes A and B (1 and 2) are the first examples of bromobisabolane sesquiterpenes possessing a phenyl moiety among Laurencia-derived sesquiterpenes, while okamuragenin (6) was the first acetogenin aldehyde possessing a C12-carbon skeleton. Each of the isolated compounds was evaluated for the brine shrimp (Artemia salina) lethal assay and 7-hydroxylaurene displayed potent lethality with LD50 1.8 µM.

Six cytotoxic annonaceous acetogenins from Annona squamosa seeds.

Custard apple (Annona squamosa L.) is an edible tropical fruit, and its seeds had been used in south China as a folk medicine to treat "malignant sore" (cancer) and as an insecticide. Phytochemical investigation of the ethanol fraction of custard apple seeds led to the isolation of six new annonaceous acetogenins: annosquacins A-D (1-4), annosquatin A (5) and annosquatin B (6). Their structures were elucidated by spectroscopic analysis. Compounds 1-4 are adjacent bistetrahydrofuran annonaceous acetogenins. Compounds 5 and 6 are non-adjacent bistetrahydrofuran annonaceous acetogenins and the first examples in which the tetrahydrofuran ring system is located between C-9 and C-20. The absolute configurations of 1-6 were defined by the application of the Mosher method. Compounds 1-6 exhibited potent cytotoxic activity in vitro against five human tumour cell lines. Compounds 5 and 6 showed a high selectivity toward the MCF-7 and A-549 cell line respectively.

Insecticidal effects of acetogenins from Rollinia occidentalis seed extract.

From the methanol seed extract of Rollinia occidentalis five acetogenins were isolated, sylvaticin (1), rolliniastatin-1 (2), rolliniastatin-2 (3), motrilin (4) and desacetyluvaricin (5), and evaluated for their toxicity and nutritional alterations on Spodoptera frugiperda Smith (Lepidoptera: Noctuidae). All but one of the compounds produced more than 90% mortality on early larval instars at 50 and 100 microg per g of diet. Sylvaticin caused 15 and 60% mortality at 50 and 100 microg/g, respectively, while the methanol seed extract reached 35 and 50% at 100 and 250 microg/g, respectively. The addition of R. occidentalis methanolic seed extract (100 and 250 microg/g) and sylvaticin (100 microg/g) to the larval diet brought about significant changes to the nutritional indices in connection with larvae fed the control diet. This is the first report on the isolation of acetogenins from R. occidentalis and their insecticidal effects on S. frugiperda.

Leishmanicidal activity and cytotoxicity of compounds from two Annonacea species cultivated in Northeastern Brazil.

INTRODUCTION: Visceral leishmaniasis is endemic in 88 countries, with a total of 12 million people infected and 350 million at risk. In the search for new leishmanicidal agents, alkaloids and acetogenins isolated from leaves of Annona squamosa and seeds of Annona muricata were tested against promastigote and amastigote forms of Leishmania chagasi. METHODS: Methanol-water (80:20) extracts of A. squamosa leaves and A. muricata seeds were extracted with 10% phosphoric acid and organic solvents to obtain the alkaloid and acetogenin-rich extracts. These extracts were chromatographed on a silica gel column and eluted with a mixture of several solvents in crescent order of polarity. The compounds were identified by spectroscopic analysis. The isolated compounds were tested against Leishmania chagasi, which is responsible for American visceral leishmaniasis, using the MTT test assay. The cytotoxicity assay was evaluated for all isolated compounds, and for this assay, RAW 264.7 cells were used. RESULTS: O-methylarmepavine, a benzylisoquinolinic alkaloid, and a C37 trihydroxy adjacent bistetrahydrofuran acetogenin were isolated from A. squamosa, while two acetogenins, annonacinone and corossolone, were isolated from A. muricata. Against promastigotes, the alkaloid showed an IC50 of 23.3 µg/mL, and the acetogenins showed an IC50 ranging from 25.9 to 37.6 µg/mL; in the amastigote assay, the IC50 values ranged from 13.5 to 28.7 µg/mL. The cytotoxicity assay showed results ranging from 43.5 to 79.9 µg/mL. CONCLUSIONS: These results characterize A. squamosa and A. muricata as potential sources of leishmanicidal agents. Plants from Annonaceae are rich sources of natural compounds and an important tool in the search for new leishmanicidal therapies.

Leishmanicidal activity and cytotoxicity of compounds from two Annonacea species cultivated in Northeastern Brazil / Atividade leishmanicida e citotoxicidade de constituintes químicos de duas espécies de Annonaceae cultivadas no nordeste do Brasil

INTRODUCTION: Visceral leishmaniasis is endemic in 88 countries, with a total of 12 million people infected and 350 million at risk. In the search for new leishmanicidal agents, alkaloids and acetogenins isolated from leaves of Annona squamosa and seeds of Annona muricata were tested against promastigote and amastigote forms of Leishmania chagasi. METHODS: Methanol-water (80:20) extracts of A. squamosa leaves and A. muricata seeds were extracted with 10 percent phosphoric acid and organic solvents to obtain the alkaloid and acetogenin-rich extracts. These extracts were chromatographed on a silica gel column and eluted with a mixture of several solvents in crescent order of polarity. The compounds were identified by spectroscopic analysis. The isolated compounds were tested against Leishmania chagasi, which is responsible for American visceral leishmaniasis, using the MTT test assay. The cytotoxicity assay was evaluated for all isolated compounds, and for this assay, RAW 264.7 cells were used. RESULTS: O-methylarmepavine, a benzylisoquinolinic alkaloid, and a C37 trihydroxy adjacent bistetrahydrofuran acetogenin were isolated from A. squamosa, while two acetogenins, annonacinone and corossolone, were isolated from A. muricata. Against promastigotes, the alkaloid showed an IC50 of 23.3 µg/mL, and the acetogenins showed an IC50 ranging from 25.9 to 37.6 µg/mL; in the amastigote assay, the IC50 values ranged from 13.5 to 28.7 µg/mL. The cytotoxicity assay showed results ranging from 43.5 to 79.9 µg/mL. CONCLUSIONS: These results characterize A. squamosa and A. muricata as potential sources of leishmanicidal agents. Plants from Annonaceae are rich sources of natural compounds and an important tool in the search for new leishmanicidal therapies.

INTRODUÇÃO: A leishmaniose visceral é uma enfermidade endêmica em 88 países, com um total de 12 milhões de pessoas infectadas e 350 milhões em risco. Na procura de novos agentes com ação leishmanicida, alcalóides e acetogeninas isoladas de Annona squamosa e Annona muricata, foram testados contra as formas promastigotas e amastigotas de Leishmania chagasi. MÉTODOS: Foram preparados extratos com metanol: água (80: 20) das folhas de A. squamosa e sementes de A. muricata que foram extraídos com solução de ácido fosfórico 10 por cento e solventes orgânicos, para obter extratos ricos em alcalóides e acetogeninas. Estes extratos foram cromatografados em coluna de sílica gel sendo eluídos com solventes de diferentes polaridades para o isolamento dos constituintes, e feita a determinação estrutural por análise espectroscópica. Os constituintes isolados foram testados contra Leishmania chagasi, responsável pela leishmaniose visceral, utilizando o teste MTT. Testes de toxicidade foram realizados em todos os compostos isolados, sendo utilizadas células RAW 264.7. RESULTADOS: Um alcalóide benzilisoquinolínico, O-metilarmepavina, e uma C37-triidróxi-acetogenina com anel bistetrahidrofurânico adjacente foram isolados de A. squamosa e duas acetogeninas annonacinona e corossolona da A. muricata. O alcalóide mostrou um índice de inibição médio (IC50) de 23,3µg/mL e as acetogeninas apresentaram IC50 variando entre 25,9 a 37,6µg/mL contra promastigotas, e no ensaio de amastigotas, o IC50 valores variaram entre 13,5 a 28,7 µg/mL. A toxicidade mostrou resultados que variaram entre 43,5 a 79,9µg/mL. CONCLUSÕES: Estes resultados caracterizam A. squamosa e A. muricata como fontes potenciais de agentes leishmanicidas.

Design, synthesis of symmetrical bivalent mimetics of annonaceous acetogenins and their cytotoxicities.

A new series of linear dimeric compounds mimicking naturally occurring annonaceous acetogenins have been synthesized by bivalent analogue design, and their cytotoxicities have been evaluated against the growth of cancer cells by MTT method. Most of these compounds show selective action favored to human cancer cell lines over normal cell lines, and compound 9 with bis-terminal benzoquinone functionality exhibits an IC(50)=0.40 µM against MCF7 cell lines. This work mentions that appropriate conformational constraints might be a useful optimizing tool for this unique class of anticancer compounds.

Insecticidal, mutagenic and genotoxic evaluation of annonaceous acetogenins.

Annonaceous acetogenins represent a class of bioactive compounds whose primary mode of action is the inhibition of NADH-ubiquinone oxidoreductase (Mitochondrial Complex I). Given the potential pesticidal use of these compounds, we evaluated the effects of seven acetogenins: squamocin (1), molvizarin (2), itrabin (3), almuñequin (4), cherimolin-1 (5), cherimolin-2 (6), and tucumanin (7) isolated from Annona cherimolia Mill. against Ceratitis capitata Wiedemann (Tephritidae). These acetogenins did not display insecticidal action at 250 microg of treatment per g of adult diet. However, the oviposition capacity of C. capitata females was significantly altered by some of the acetogenins at this concentration. The most potent compounds were itrabin, molvizarin and squamocin. Moreover, significant differences were detected in the preference of oviposition sites when itrabin and squamocin were spread on the surface of artificial fruits at doses of 30 microg/cm2. Additionally, we investigated the mutagenic effects displayed by itrabin, as well as the phytotoxic and genotoxic action of squamocin and itrabin. Both compounds displayed slight phytotoxic and genotoxic effects on roots of Allium cepa at 2.5 microg/mL though no mutagenic effects were detected at 0.25, 0.5 and 2.5 microg/mL on Salmonella typhimurium strains TA98 and TA100.

Acetogenin and prenylated flavonoids from Helminthostachys zeylanica with inhibitory activity on superoxide generation and elastase release by neutrophils.

One new acetogenin, 6-hydroxy-8-pentadecyloxocane-2,7-dione ( 1), and four new prenylated flavonoids, 4''a,5'',6'',7'',8'',8''a-hexahydro-5,3',4'-trihydroxy-5'',5'',8''a-trimethyl-4 H-chromeno[2'',3'':7,6]flavone ( 2), 4''a,5'',6'',7'',8'',8''a-hexahydro-5,3',4',-trihydroxy-5'',5'',8''a-trimethyl-4 H-chromeno[2'',3'':7,8]flavone ( 3), 2-(3,4-dihydroxyphenyl)-6-((2,2-dimethyl-6-methylenecyclohexyl)methyl)-5,7-dihydroxy-chroman-4-one ( 4), and 2-(3,4-dihydroxy-2-[(2,6,6-trimethylcyclohex-2-enyl)methyl]phenyl)-3,5,7-trihydroxy-4 H-chromen-4-one ( 5), together with six known compounds, were isolated and purified from the rhizomes of Helminthostachys zeylanica by column chromatography and high performance liquid chromatography (HPLC) via bioactivity-guided fractionation isolation. The structures of the new isolates were elucidated by spectroscopic methods. Compounds 1, 3, and 5 showed inhibitory activities on either superoxide anion generation or elastase release by human neutrophils in response to formyl-L-methionyl-L-leucyl-L-phenylalanine/cytochalasin B (FMLP/CB).

Genotoxic and cytotoxic effects produced by acetogenins obtained from Annona cherimolia Mill.

In the present study we extracted three isomeric acetogenins (Ace) from the seeds of Annona cherimolia Mill. (Annonaceae) and determined their genotoxic and cytotoxic potential in mice. Our results showed a significant increase in the rate of micronucleated polychromatic erythrocytes induced by Ace with respect to the value for the control group; the effect was less pronounced than that observed with daunorubicin (Dau). To evaluate cytotoxicity, we determined the proportion of polychromatic erythrocytes with respect to the number of normochromatic erythrocytes, and we found an inhibitory effect induced by Ace that was quite similar to that observed with Dau. Besides, by means of the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide test, we also determined the cytotoxicity of Ace in both a normal fibroblast mouse cell line and in a line derived from human colon cancer. In this assay, the strongest decrease in viability corresponded to the malignant cell line. Our results established for the first time the genotoxic capacity of these compounds in vivo, and confirmed their cytotoxic potential in cultured cells.